title: Sample 11
description: Protocols: Cells were transfected at 80% confluence using a mixture of siLenfect (Bio-Rad Laboratories AB, Solna, Sweden) and siRNA (20 nM), according to the manufacturer (Dharmacon/GE Healthcare, Uppsala, Sweden). Forty-eight hours after transfection cells were either stimulated with 5ng/ml TGFβ or 30ng/ml BMP7, or were unstimulated as control. The patient-derived glioblastoma cells U3034MG were cultured in adherent conditions on laminin (Sigma-Aldrich Sweden AB, Stockholm, Sweden), in DMEM/F12 Glutamax (GIBCO, Life Technologies Europe BV, Stockholm, Sweden) and Neurobasal medium (GIBCO, Life Technologies Europe BV, Stockholm, Sweden) mixed in 1:1 ratio, with the addition of 1% B27 (Invitrogen, Life Technologies Europe BV), 1% N2 (Invitrogen, Life Technologies Europe BV), 1% penicillin/streptomycin (Sigma-Aldrich Sweden AB, Stockholm, Sweden), 10 ng/ml EGF and FGF2 (PeproTech, EC Ltd, London, UK). Cellular RNA from ~250,000 cells was purified with the Nucleospin RNA kit ((Macherey-Nagel, Düren, Germany). RNA concentration was measured with ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA for Ampliseq was extracted from 3 biological replicates as described above. Fifty ng of RNA was reverse transcribed to cDNA using the Ion AmpliSeq Transcriptome Human Gene Expression Kit Preparation protocol (Revision A.0, Life Technologies). The acquired cDNA was amplified using Ion AmpliSeq Transcriptome Human Gene Expression core panel (Life Technologies) and the primer sequences were then partially digested. Then, DNA adaptors (Ion P1 Adaptor and Ion Xpress Barcode Adaptor, Life Technologies) were ligated to the amplicons. Adaptor ligated amplicons were purified using the Agencourt AMPure XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix, Life Technologies) and amplified. Size-selection and purification was conducted using Agencourt AMPure XP reagent (Beckman Coulter).
attributes.tag: ENA first public
attributes.value: 2021-05-12
attributes.tag: ENA last update
attributes.value: 2021-05-12
attributes.tag: External Id
attributes.value: SAMEA7625092
attributes.tag: INSDC center name
attributes.value: UU
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:53Z
attributes.tag: INSDC last update
attributes.value: 2020-11-25T14:57:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9813:Sample 11
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: U3034MG
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: brain glioblastoma
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: brain
attributes.tag: replicate
attributes.value: 2
attributes.tag: sample name
attributes.value: E-MTAB-9813:Sample 11